A:The use of ChIP validated antibodies is essential for the success of a ChIP assay. The antibody must recognise and bind to native protein that is bound to DNA. It is essential to include ChIP validated positive and negative antibody controls to ensure chromatin preparation and ChIP methodology are appropriate. Antibodies from other applications do not always work well in ChIP. The Chromatrap® premium kit is supplied with positive and negative control antibodies to ensure the efficiency of the IP step.
The antibody epitope may have been destroyed or masked during the cross-linking process or it may be hidden by other proteins in the chromatin complex. Reduce the cross-linking incubation time or find an antibody which has alternative epitope.There may be an incorrect addition of antibody to chromatin sample, carryout an antibody dilution series to determine the optimum ratio. Optimal results have been achieved with a 2:1 antibody: chromatin ratio for qPCR and 30 µg chromatin and 5 µg antibody for sequencing in the Chromatrap® spin columns.
The antibody may be of low affinity and may require a longer incubation time than that suggested in the protocol, increase the ChIP reaction to an overnight incubation at 4°C.
Chromatin may be over or under sheared. Optimal cross-linking of DNA with proteins ensures that the chromatin structure is preserved during the isolation and ChIP procedure. The optimal time for crosslinking will vary with cell line. Too little cross-linking will result in DNA and antigenic protein loss. Lower formaldehyde concentrations or shorter incubation times may improve shearing efficiency but in doing so may result in less efficient cross-linking, reducing the yield of precipitated chromatin. Longer incubation times can result in over cross-linking. Chromatin becomes over cross-linked when it is impossible to recover a substantial amount by reverse cross-linking. Over cross-linking results in elevated background and reduced antigen availability.
The kit supplied primers recognise the promoter region of the human GAPDH gene. If you are using chromatin from a non human source (eg. Mouse) appropriate control primers will need to be designed.
A selection of publications using Chromatrap in peer reviewed journals can be found here.
Chromatrap ChIP-seq kits are compatible with mass spectroscopy as a downstream process. Instructions on how to prepare samples can be found in the relevant protocols.
Chromatrap ChIP-seq kits and Premium qPCR kits come complete with a control antibody and primer set to ensure you have the best quality chromatin for ChIP. Control antibodies and primer sets for verification of your samples are also available from the Chromatrap range.